Methods for producing mouse and human endostatin are disclosed. Methods for refolding and purifying endostatin from inclusion bodies expressed in bacteria and nucleic acids encoding full-length and truncated forms of endostatin are also disclosed.
Angiogenesis, the growth of new blood vessels, plays an important role in cancer growth and metastasis. In humans, the extent of vasculature in a tumor has been shown to correlate with the patient prognosis for a variety of cancers (Folkman, J., Seminars in Medicine of the Beth Israel Hospital, Boston 333(26): 1757-1763, 1995; Gasparini, G., European Journal of Cancer 32A(14): 2485-2493, 1996; Pluda, J. M., Seminars in Oncology 24(2): 203-218, 1997; Norrby, K, APMIS 105: 417-437, 1997). In normal adults, angiogenesis is limited to well controlled situations, such as wound healing and the female reproductive system (Battegay, E. J., J Mol Med 73:-333-346, 1995; Dvorak, H. F, New Engl J Med, 315: 1650-1659, 1986).
Animal studies suggest that tumors can exist in a dormant state, in which tumor growth is limited by a balance between high rates of proliferation and high rates of apoptosis (Holmgren, L. et al., Nat. Med. (N. Y.) 1(2): 149-153, 1995; Hanahan, D. et al., Cell 86(3): 353-364, 1996). The switch to an angiogenic phenotype allows tumor cells to escape from dormancy and to grow rapidly, presumably as the result of a decrease in the apoptotic rate of the tumor cells (Bouck, Cancer Cells, 2(6): 179-185, 1990; Dameron et al, Cold Spring Harb Symp Quant Biol, 59: 483-489, 1994). The control of angiogenesis is thought to be a balance between factors which promote new vessel formation and anti-angiogenic factors with suppress the formation of a neovasculature (Bouck, N. et al., Advances in Cancer Research 69: 135-173, 1996; O""Reilly et al., Cell 79(2): 315-328, 1994).
A variety of pro-angiogenic factors have been characterized including basic and acid fibroblast growth factors (bFGF and aFGF) and vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) (Potgens, A. J. G. et al., Biol. Chem. Hoppe-Seyler 376: 57-70, 1995; Ferrara, N., European Journal of Cancer 32A(14): 2413-2442, 1996; Bikfalvi, A. et al., Endocrine Reviews 18: 26-45, 1997). Several endogenous anti-angiogenic factors have also been characterized, including angiostatin (O""Reilly et al., Cell 79(2): 315-328, 1994), endostatin (O""Reilly et al, Cell 88(2): 277-285, 1997), interferon-alpha. (Ezekowitz et al, N. Engl. J. Med., May 28, 326(22) 1456-1463, 1992), thrombospondin (Good et al, Proc Natl Acad Sci USA 87(17): 6624-6628, 1990; Tolsma et al., J Cell Biol 122(2): 497-511, 1993), and platelet factor 4 (PF4) (Maione et al, Science 247(4938): 77-79, 1990).
Many angiogenic inhibitors are in clinical development (See Shawver et al., Drug Discovery Today 2(2): 50-63, 1997, and references therein). Polypeptides such as interferon alpha and platelet factor 4 are in clinical trials. Angiostatin, soluble Flt-1 receptor, and bactericidal/permeability increasing protein derivative 23 are in preclinical studies. Monoclonal antibodies such as humanized anti-avb3 antibody (LM609), anti-VEGF, and anti-Flk-1 monoclonal antibody (DC101) are also in preclinical studies. Tecogalan (DS4152), a sulfated polysaccharide-peptidoglycan complex is in clinical trials, and bFGF carbohydrate inhibitor (GM1474) and glyceptor mimetic inhibitor of bFGF (GL14.2) are in preclinical studies. The antibiotic AGM1470 (TNP470), a fumagillin analog, and Suramin, a polyanionic compound are in clinical trials. Small molecule inhibitors such as urokinase receptor antagonists, inhibitors of phospatidic acid, inhibitors of Flk-1, and inhibitors of VEGF-F11 binding are all in preclinical studies. Thalidomide, and its analogues, and matrix metalloproteinase inhibitors, such as Batimastat/Marimastat, are in clinical trials. Oligonucleotides, such as ribozymes that target VEGF receptors and VEGF anti-sense oligonucleotides, are also in preclinical trials.
Anti-angiogenic therapy may offer several advantages over conventional chemotherapy for the treatment of cancer. Anti-angiogenic agents have low toxicity in preclinical trials and development of drug resistance has not been observed (Folkman, J., Seminars in Medicine of the Beth Israel Hospital, Boston 333(26): 1757-1763, 1995). As angiogenesis is a complex process, made up of many steps including invasion, proliferation and migration of endothelial cells, it can be anticipated that combination therapies may be most effective. In fact, combinations of chemotherapy with anti-angiogenic therapy have already shown promising results in pre-clinical models (Teicher, B. A. et al., Breast Cancer Research and Treatment 36: 227-236, 1995; Teicher, B. A. et al., European Journal of Cancer 32A(14): 2461-2466, 1996).
Endostatin is a 20 kDa protein derived from the C-terminal fragment of alpha 1 type collagen XVIII. Conditioned cell culture media from a hemangioendothelioma cell line (EOMA) was shown to contain a factor which inhibited endothelial cell proliferation in vitro (O""Reilly et al., Cell 88: 277-285, 1997). The factor responsible for this inhibition was named endostatin. A recombinant form of this protein expressed in baculovirus-infected insect cells inhibited the growth of metastases in the Lewis lung tumor model and an insoluble E. coli derived form of this protein was shown to be efficacious in preventing primary tumor growth in several tumor models (O""Reilly et al., Cell 88: 277-285, 1997; Boehm et al., Nature 390: 404-410, 1997).
Although many types of expression systems have been developed over the past twenty years, bacterial systems, particularly those based on E. coli, are widely used for the production of proteins on an industrial scale. Vectors which permit high level expression and the ability to carry out fermentations at high cell densities and low cost, have contributed to the extensive development and use of E. coli-based expression systems. One significant problem, however, is the tendency of E. coli to form inclusion bodies which contain the desired recombinant protein. Inclusion body formation necessitates additional downstream processing, such as in vitro refolding, before biologically active proteins can be recovered. The tendency to form insoluble aggregates does not appear to correlate with factors such as size, hydrophobicity, subunit structure, or the use of fusion domains (Kane J. F. and Harley, D. L., Tibtech 6: 95, 1988). Inclusion body formation appears to be determined by the rates of protein synthesis, folding, aggregation, and proteolytic degradation, the solubility and thermodynamics of folding intermediates and native proteins, and the interactions of these species with chaperone proteins (Rainer Rudolph, In Protein Engineering: Principles and Practice, Edited by Jeffrey L. Cleland and Charles S. Craik, p 283-298, Wiley-Liss, Inc., New York, N.Y., 1996).
Inclusion bodies generally form in the cytoplasm of cells expressing a recombinant protein at high levels. They refract light when observed by phase contrast microscopy and thus are sometimes referred to as refractile bodies. The inclusion bodies are characterized by a relatively high specific density and can be pelleted from lysed cells by centrifugation. The formation of inclusion bodies may protect recombinant proteins from proteolysis as they do not easily disintegrate under physiological solvent conditions. High concentrations of denaturants, such as 6 M guanidine hydrochloride or 6-8 M urea, have been commonly used to solubilize the proteins present in inclusion bodies. A variety of inclusion body solubilization protocols have been compared (Fisher, B., Summer, L. and Goodenough, P. Biotechnol. Bioeng. 1: 3-13, 1992).
Although the desired foreign gene product is the main component of inclusion bodies, other host cell proteins such as small heat shock proteins, outer membrane proteins, elongation factor EF-TU, and RNA polymerase may also be enriched in such preparations (Allen, S. P., Polazzi, J. O. Gierse, J. K., and Easton, A. M., J. Bacteriol. 174: 6938-6947, 1992); Hart, R. A., Rinas, U., and Bailey, J. E., J. Biol. Chem. 265: 12728-12733, 1990; Hartley, D. L., and Kane, J. F., Biochem. Soc. Trans. 16: 101, 1988).
World patent application WO 97/15666 describes the expression, purification, and characterization of endostatin from E. coli and baculovirus-infected insect cells. The bacterially-derived endostatin was not refolded into its native state, but was utilized as an insoluble suspension for most of these studies. This disclosure also describes the purification of native endostatin from conditioned media of the murine hemangioendothelioma cell line EOMA. Endostatin was purified from this conditioned media through classical purification methodology.
No successful attempts at refolding endostatin have been described (O""Reilly et al., Cell 88: 277-285, 1997). The E. coli-derived recombinant endostatin characterized by these authors precipitated following dialysis against PBS. The precipitated (nonrefolded) material could not be tested in vitro because of its insolubility in culture media. A small (unspecified) percentage of the material spontaneously solubilized in the PBS during dialysis. This material had comparable inhibitory activity in endothelial cell activities as both native and soluble baculovirus-derived endostatin. When the E. coli-derived recombinant endostatin was refolded in the presence of 0.1 M sodium phosphate, pH 7.4, 150 mM NaCl, 0.6 M urea, 2 mM reduced glutathione, 0.02 mM oxidized glutathione, and 0.5 M arginine at a final concentration of 0.1 mg/ml, over 99% of the protein was lost. This great loss precluded use of this material for in vivo assays. Instead, the authors used the uncharacterized insoluble (nonrefolded) form of endostatin for most of their in vivo studies. The E. coli-derived endostatin precipitate was observed to dissolve gradually over five days and produce a sustained anti-angiogenic effect in chick chorioallantoic membrane (CAM) assays. A suspension of the same material formed a pellet at the site of injection in mice, which resorbed slowly over a 24-48 hour period.
Subsequent studies by the same group demonstrated that drug resistance does not develop when mice bearing lung carcinoma, T241 fibrosarcoma, or B16F10 melanoma are treated with mouse endostatin (Boehm, T., et al., Nature 390: 404-407, 1997). E. coli-derived recombinant murine endostatin was prepared as described earlier (O""Reilly et al., Cell 88: 277-285, 1997) except that bacteria were pelleted and resuspended in 8 M urea, 10 mM beta mercaptoethanol, and 10 mM pH 8.0, and incubated for 1 to 2 hours. Beta mercaptoethanol was eliminated in subsequent steps. Recombinant mouse endostatin was delivered to mice as a suspension in PBS. Mice bearing one of the three tumor types were injected with the purified but poorly soluble endostatin suspension into the subcutaneous dorsa at a site remote from the inoculated tumors. Treatment was stopped when tumors regressed, then allowed to regrow. Tumor growth did not recur after 6, 4, or 2 endostatin treatment cycles, respectfully, when therapy was ended.
Recently, the circulating form of human endostatin was isolated and characterized (Standker et al., FEBS Letters 420: 129-133, 1997). High molecular weight peptides (1-20 kDa) were isolated from 2,500 liters of human blood ultrafiltrate (hemofiltrate, HF) obtained from patients with chronic renal insufficiency. Extracts were bound to a preparative cation exchange column and eluted by pH pool fractionation (7 buffers with pH increasing from 3.6 to 9.0). High molecular weigh peptides were detected in pool 8, which was eluted with water, and subsequently purified by reversed-phase HPLC. Aliquots were subjected to matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and the exact molecular mass determined by electrospray mass spectroscopy (ES-MS) was found to be 18,494 Da. Cysteine residues 1-3 and 2-4 in the molecule were found to be linked by disulfide bridges. The final recovery during purification was estimated to be in the range of 20%, resulting in a concentration of  greater than 10xe2x88x9211 M in hemofiltrate. The resulting concentration of endostatin in patient plasma was estimated to be in the range of 10xe2x88x9210 M or higher. It is not known if pool of tissue bound endostatin exists, as was proposed for angiostatin (Kost et al., Eur J. Biochem. 236: 682-688, 1996). In vitro biological characterization of the native human endostatin (which was 12 amino acids shorter than mouse endostatin) showed no anti-proliferative activity on different endothelial cell types. The characterization of recombinant forms of human endostatin was not reported. The authors speculate that differences in reported activity of mouse and human forms of endostatin could be due to several factors: (i) the two forms may were isolated from different sources and may have different selectivity, specificity, or efficacy in in vitro and in vivo assays. (ii) Differences in post-translational modifications found on the peptides may account for the discrepancy in reported activities. (iii) Human endostatin may not necessarily inhibit proliferation of endothelial cells, but indirectly influence other cellular components which are observable only in a complex in vivo system.
One object of the invention is to describe a method for expressing high levels of endostatin in bacteria.
Another aspect of the invention is to describe an efficient method by which endostatin inclusion bodies can be solubilized, subsequently refolded, and purified to generate biologically-active material.
Preferably the steps of solubilizing mouse or human endostatin inclusion bodies are carried out at an elevated pH. Preferably the elevated pH in the solubilizing step is carried out at a pH ranging from about pH 9 to about pH 11.5. Even more preferably the elevated pH range is from about pH 10 to about pH 11. Most preferably, the elevated pH is about 10.5.
Preferably the steps of refolding mouse or human endostatin inclusion bodies are carried out at a near-neutral pH. Preferably the near-neutral pH in the solubilizing step is carried out at a pH ranging from about pH 6 to about pH 8.5. Even more preferably the near-neutral pH range for refolding mouse endostatin is from about pH 7.0 to about pH 8.0. Most preferably, near-neutral pH range for refolding mouse endostatin is about pH 7.5. Even more preferably the near-neutral pH range for refolding human endostatin is from about pH 7.0 to about pH 8.0. Most preferably, near-neutral pH range for refolding human endostatin is about pH 7.5.
Preferably, the concentration of endostatin gene product is present at a concentration of from about 0.2 to about 20 mg/ml during the solubilization step. Even more preferably, the concentration is about 2.5 mg/ml.
Preferably, the concentration of endostatin gene product is present at a concentration of from about 0.02 to about 2 mg/ml during the refolding step. Even more preferably, the concentration is about 0.25 mg/ml.
Preferably a denaturant selected from urea and guanidine hydrochloride is used during the solubilization and refolding steps. Even more preferably, the denaturant is urea.
Preferably the concentration of urea is from about 4 M to about 10 M during the solubilization step. Even more preferably, the concentration is about 6 M.
Preferably the concentration of urea is from about 2 M to about 4 M during the refolding step. Even more preferably, the concentration is about 3.5 M.
Preferably the concentration of guanidine hydrochloride is from about 2 M to about 8 M during the solubilization step. Even more preferably, the concentration is about 4 M.
Preferably the concentration of guanidine hydrochloride is from about 0.2 M to about 2 M during the refolding step. Even more preferably, the concentration is about 1.5 M.
Preferably, the solubilization and reducing steps are carried out in the presence of a reducing agent capable of reducing disulfide linkages to sulfhydryl groups. Preferably the reducing agent is selected from the group consisting of DTT, BME, cysteine, and reduced glutathione. Even more preferably, the reducing agent is DTT or cysteine.
Preferably, DTT is present at a concentration of from about 2 mM to about 10 mM during the solubilization step. Even more preferably, the concentration is about 5 mM.
Preferably, DTT is present at a concentration of from about 0.5 mM to about 2 mM during the refolding step. Even more preferably, the concentration is about 0.5 mM.
Preferably, reduced glutathione is present at a concentration of from about 5 mM to about 20 mM during the solubilization step. Even more preferably, the concentration is about 10 mM.
Preferably, reduced glutathione is present at a concentration of from about 0.5 mM to about 4 mM during the refolding step. Even more preferably, the concentration is about 1 mM.
Preferably, cysteine is present at a concentration of from about 5 mM to about 20 mM during the solubilization step. Even more preferably, the concentration is about 10 mM.
Preferably, cysteine is present at a concentration of from about 0.5 mM to about 4 mM during the refolding step. Even more preferably, the concentration is about 1 mM.
Preferably, an agent capable of enhancing the interchange of disulfide bonds is present during the refolding step. Preferably the agent is selected from cystine and oxidized glutathione. Even more preferably, the agent is cystine.
Preferably, cystine is present at a concentration of from about 0.2 mM to about 5 mM during the refolding step. Even more preferably, the concentration is about 1 mM.
Preferably disulfide bonds are formed through air oxidation during the refolding step. Preferably, the air oxidation step is carried out from about 12 to about 96 hours. Even more preferably, the air oxidation step is carried out from about 24 to about 72 hours. Most preferably the air oxidation step is carried out about 60 hours.
Preferably refolded endostatin is further purified by a process selected from but not limited to the group consisting of ion-exchange chromatography, hydrophobic interaction chromatography and RP-HPLC.
Preferably the method of expression, solubilization, refolding and purification uses endostatin genes that are either mouse or human. Even more preferably these genes are selected from the group consisting of SEQ ID NOs: 5-9.
Novel proteins of this invention are modified human or mouse endostatin amino acid sequences, and said protein can optionally be immediately preceded by (methioninexe2x88x921), (alaninexe2x88x921), (methioninexe2x88x922, alaninexe2x88x921), (serinexe2x88x921), (methioninexe2x88x922, serinexe2x88x921), (cysteinexe2x88x921), or (methioninexe2x88x922, cysteinexe2x88x921).
Additionally, the present invention relates to recombinant expression vectors comprising nucleotide sequences encoding endostatin, variants and muteins of endostatin, related microbial and eukaryotic expression systems, and processes for making (comprising the steps of expressing, solubilizing, refolding, purifying) these proteins.
Cloning of DNA sequences encoding these proteins may be accomplished by the use of intermediate vectors. Alternatively, one gene can be cloned directly into a vector containing the other gene. Linkers and adapters can be used for joining the DNA sequences, as well as replacing lost sequences, where a restriction site was internal to the region of interest. Thus genetic material (DNA) encoding one polypeptide, peptide linker, and the other polypeptide is inserted into a suitable expression vector which is used to transform bacteria, yeast, insect cells or mammalian cells. The transformed organism or cell line is grown and the protein isolated by standard techniques. The resulting product is therefore a new protein which has all or a portion of one protein joined by a linker region to all or a portion of second protein.
Another aspect of the present invention includes plasmid DNA vectors for use in the expression of these proteins. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Appropriate vectors which can transform microorganisms or cell lines capable of expressing the proteins include expression vectors comprising nucleotide sequences coding for the proteins joined to transcriptional and translational regulatory sequences which are selected according to the host cells used.
Vectors incorporating modified sequences as described above are included in the present invention and are useful in the production of the proteins. The vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention and which are capable of directing the replication and expression thereof in selected host cells.
Methods for producing these proteins is another aspect of the present invention. The method of the present invention involves culturing suitable cells or cell lines, which has been transformed with a vector containing a DNA sequence coding for expression of a novel multi-functional protein. Suitable cells or cell lines may be bacterial cells. For. example, the various strains of E. coli are well-known as host cells in the field of biotechnology. Examples of such strains include E. coli strains JM101 (Yanisch-Perron et al. Gene 33: 103-119, 1985) and MON105 (Obukowicz et al., Applied Environmental Microbiology 58: 1511-1523, 1992). Also included in the present invention is the expression of the multi-functional proteins utilizing a chromosomal expression vector for E. coli based on the bacteriophage Mu (Weinberg et al., Gene 126: 25-33, 1993). Various strains of B. subtilis may also be employed in this method. Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention.
When expressed in the E. coli cytoplasm, the gene encoding the proteins of the present invention may also be constructed such that at the 5xe2x80x2 end of the gene codons are added to encode Metxe2x88x922-Alaxe2x88x921, Metxe2x88x922-Serxe2x88x921, Metxe2x88x922-Cysxe2x88x921, or Metxe2x88x921 at the N-terminus of the protein. The N termini of proteins made in the cytoplasm of E. coli are affected by post-translational processing by methionine aminopeptidase (Ben Bassat et al., J. Bacteriol. 169:751-757, 1987) and possibly by other peptidases so that upon expression the methionine is cleaved off the N-terminus. The proteins of the present invention may include polypeptides having Metxe2x88x921, Alaxe2x88x921, Serxe2x88x921, Cysxe2x88x921, Metxe2x88x922-Alaxe2x88x921, Metxe2x88x922-Serxe2x88x921, or Metxe2x88x922-Cysxe2x88x921 at the N-terminus. These mutant proteins may also be expressed in E. coli by fusing a secretion signal peptide to the N-terminus. This signal peptide is cleaved from the polypeptide as part of the secretion process.
The following is a list of abbreviations and the corresponding meanings as used interchangeably herein:
g=gram(s)
HPLC=high performance liquid chromatography
mg=milligram
ml=milliliter
DTT=dithiothreitol
RT=room temperature
PBS=phosphate buffered saline
The following is a list of definitions of various terms used herein:
The term xe2x80x9canti-tumorxe2x80x9d means possessing an activity which slows or abolishes the growth of, or which kills, or otherwise harms tumors in vivo.
The term xe2x80x9cnative sequencexe2x80x9d refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein.
The terms xe2x80x9cmutant amino acid sequence,xe2x80x9d xe2x80x9cmutant proteinxe2x80x9d, xe2x80x9cvariant proteinxe2x80x9d, xe2x80x9cmuteinxe2x80x9d, or xe2x80x9cmutant polypeptidexe2x80x9d refer to a polypeptide having an amino acid sequence which varies from a native sequence due to amino acid additions, deletions, substitutions, or any combination thereof, or is encoded by a nucleotide sequence from an intentionally-made variant derived from a native sequence or chemically synthesized.
The term xe2x80x9cendostatinxe2x80x9d means a protein fragment of collagen XVIII having anti-angiogenic activity. The activity of said fragments can be determined by the mouse corneal micropocket assay of angiogenesis or by inhibition of endothelial cell growth or migration in vitro. Preferably, mouse endostatin means the sequence depicted in SEQ ID 10, and human endostatin means the sequence depicted in SEQ ID NO 11.